Spatial Patterns in Hyphal Growth and Substrate Exploitation within Norway Spruce Stems Colonized by the Pathogenic White-Rot Fungus Heterobasidion parviporum

In Norway spruce, a fungistatic reaction zone with a high pH and enrichment of phenolics is formed in the sapwood facing heartwood colonized by the white-rot fungus Heterobasidion parviporum. Fungal penetration of the reaction zone eventually results in expansion of this xylem defense. To obtain information about mechanisms operating upon heartwood and reaction zone colonization by the pathogen, hyphal growth and wood degradation were investigated using real-time PCR, microscopy, and comparative wood density analysis of naturally colonized trees with extensive stem decay. The hyphae associated with delignified wood at stump level were devoid of any extracellular matrix, whereas incipient decay at the top of decay columns was characterized by a carbohydrate-rich hyphal sheath attaching hyphae to tracheid walls. The amount of pathogen DNA peaked in aniline wood, a narrow darkened tissue at the colony border apparently representing a compromised region of the reaction zone. Vigorous production of pathogen conidiophores occurred in this region. Colonization of aniline wood was characterized by hyphal growth within polyphenolic lumen deposits in tracheids and rays, and the hyphae were fully encased in a carbohydrate-rich extracellular matrix. Together, these data indicate that the interaction of the fungus with the reaction zone involves a local concentration of fungal biomass that forms an efficient translocation channel for nutrients. Finally, the enhanced production of the hyphal sheath may be instrumental in lateral expansion of the decay column beyond the reaction zone boundary.To grow to great heights, trees continually replace their water- and nutrient-conducting elements. Older elements, such as the heartwood that is formed in many trees, gradually become nonconductive. In contrast to the living sapwood, heartwood lacks active defense mechanisms against microbes. However, lignin, the polymer coating cell wall polysaccharides, is highly resistant to microbial degradation. In fact, white-rot fungi, besides having evolved the ability to tolerate or detoxify the secondary metabolites accumulating in heartwood, are the only organisms capable of efficiently degrading lignin. Following establishment in the heartwood of living trees, the colonies of pathogenic white-rot fungi expand and eventually also threaten the conductive sapwood.The white-rot fungus Heterobasidion annosum sensu lato, composed of three species with overlapping geographic distributions and host ranges in Europe (23), is the most important pathogen of Norway spruce (Picea abies L. Karst) in boreal forests. Primary infection of Norway spruce stands by H. annosum sensu lato takes place through fresh thinning stumps or wounds on roots and at the base of the stem. Basidiospores landing on these entrance points give rise to mycelia which colonize the root systems, and eventually the fungus spreads into the stem heartwood. At sites infested with Heterobasidion parviporum, a species primarily restricted to Norway spruce, roots of saplings can become infected by the fungus after around 10 years of growth (25). Stem colonization usually initiates only after the heartwood has started to develop, which in Norway spruce takes place in trees 25 to 40 years old (17). Due to relatively rapid axial spread within heartwood, the decay column caused by H. annosum sensu lato often is up to 10 m high in the stems of mature Norway spruce trees.In response to sapwood challenge by an expanding heartwood-based colony of H. annosum sensu lato, Norway spruce forms a so-called reaction zone (RZ) in the border area between healthy sapwood and colonized heartwood. This xylem defense is characterized by high pH due to increased carbonate content and enrichment of phenolic compounds, particularly lignans, some of which have shown antifungal properties in bioassays (14, 30, 31). Although several wood decay fungi are able to eventually penetrate the RZ regions formed in trees, the strategies employed by fungi to breach these unique defense barriers are poorly understood (24). The purpose of this study was to obtain information about the mechanisms operating in heartwood colonization and expansion of the decay column via penetration of the RZ. To do this, we examined spatial growth of H. parviporum and the associated substrate exploitation patterns within naturally colonized mature stems of Norway spruce.